which is the best approach for simultaneous siRNA of two genes? - (Nov/17/2006 )

Dear all
I want to knock down two genes (A&B)simultaneously, practically speaking, I am thinking of 2 approaches;
1) diluting siRNA of gene A in OPTIMEM, diluting oligofectamine in separate volume of OPTIMEM, then after 10 minutes combine diluted oligofectamine with diluted siRNA of gene A (SOLUTION#1). similarly at the same time do the same thing for siRNA of gene B (SOLUTION#2). after 20 minutes add SOLUTION#1 (of geneA) to the cell culture, mix gently, then add SOLUTION#2 (of geneB) to the cell culture and mix gently.
2)combine siRNA for both genes A&B and then dilute in OPTIMEM , dilute oligofectamine in separate volume of OPTIMEM, then after 10 minutes combine diluted oligofectamine with diluted combined siRNA of genes A&B. after 20 minutes add to the cell culture. so in this approach siRNA for both genes are dealt with as if they are for one gene after the initial combining step.

which of these approaches is the best?

one more question, if I used 50nM final concentration of each geneA&B, what should be the final concentration of the control oligonucleotides (LacZ in my case). should it be 50nM or 100nM?

thanks alot

For your dilutions, i think the method of preparing mix of A and B separately and then plate them on cells is better, for minimize the potentiality of siRNA ointeractions which may disturb lipid encapsulation.
you should do the 2 controls.
First one for 50mM control and the second one for checking if adding 2x conc of siRNA is not toxic or does not activate others effects in cells.

I tried both approaches and I got comparable results (good knockdown in both)